Sibjotang Protects against Cardiac Hypertrophy In Vitro and In Vivo.

Cardiac hypertrophy is developed by various diseases such as myocardial infarction, valve diseases, hypertension, and aortic stenosis. Sibjotang (, Shizaotang, SJT), a classic formula in Korean traditional medicine, has been shown to modulate the equilibrium of body fluids and blood pressure. This research study sought to explore the impact and underlying process of Sibjotang on cardiotoxicity induced by DOX in H9c2 cells. In vitro, H9c2 cells were induced by DOX (1 µM) in the presence or absence of SJT (1-5 µg/mL) and incubated for 24 h. In vivo, SJT was administrated to isoproterenol (ISO)-induced cardiac hypertrophy mice (n = 8) at 100 mg/kg/day concentrations. Immunofluorescence staining revealed that SJT mitigated the enlargement of H9c2 cells caused by DOX in a dose-dependent way. Using SJT as a pretreatment notably suppressed the rise in cardiac hypertrophic marker levels induced by DOX. SJT inhibited the DOX-induced ERK1/2 and p38 MAPK signaling pathways. In addition, SJT significantly decreased the expression of the hypertrophy-associated transcription factor GATA binding factor 4 (GATA 4) induced by DOX. SJT also decreased hypertrophy-associated calcineurin and NFAT protein levels. Pretreatment with SJT significantly attenuated DOX-induced apoptosis-associated proteins such as Bax, caspase-3, and caspase-9 without affecting cell viability. In addition, the results of the in vivo study indicated that SJT significantly reduced the left ventricle/body weight ratio level. Administration of SJT reduced the expression of hypertrophy markers, such as ANP and BNP. These results suggest that SJT attenuates cardiac hypertrophy and heart failure induced by DOX or ISO through the inhibition of the calcineurin/NFAT/GATA4 pathway. Therefore, SJT may be a potential treatment for the prevention and treatment of cardiac hypertrophy that leads to heart failure.

Diesel vehicles-derived PM2.5 induces lung and cardiovascular injury attenuates by Securiniga suffruticosa: Involvement of NF-κB-mediated NLRP3 inflammasome activation pathway.

Respiratory exposure to Particulate matter (PM), including Diesel exhaust particulate (DEP), causes oxidative stress-induced lung inflammation. Especially, fine particulate matter with an aerodynamic diameter less than 2.5 µm (PM2.5) is a serious air pollutant associated with various health problems including cardiovascular diseases. The present study aimed to examine the inhibitory effect of Securiniga suffruticosa (S. suffruiticosa) on DEP and PM-induced lung and cardiovascular diseases. Mice inhaled DEP by using nebulizer chamber for two weeks. Treatment with S. suffruiticosa reduced the expression of C-X-C motif ligand 1/2 in bronchoalveolar lavage fluid and Muc5ac, ICAM-1, TNF-⍺, IL-6 mRNA in lung were also attenuated by S. suffruiticosa. In thoracic aorta, DEP increased CAMs, TNF-⍺ and inflammasome markers such as NLRP3, Caspase-1, and ASC. However, S. suffruiticosa suppressed these levels. S. suffruiticosa inhibited PM2.5 induced production of intracellular reactive oxygen species (ROS); and inhibited the translocation of NF-κB p65 to the nucleus in human umbilical vein endothelial cells. Taken together, this study proved that exposure to PM2.5 induced both lung and vascular inflammation, however, S. suffruiticosa attenuated this injury via the downregulation of the NLRP3 signaling pathway. These findings suggest that S. suffruiticosa may have potential therapeutic benefit against air pollution-mediated lung and cardiovascular diseases.

Sinkihwan-gamibang ameliorates puromycin aminonucleoside-induced nephrotic syndrome.

Nephrotic syndrome (NS) is a kidney disease characterized by hypertriglyceridemia, massive proteinuria, hypo-albuminemia and peripheral edema. Sinkihwan-gamibang (SKHGMB) was recorded in a traditional Chinese medical book named "Bangyakhappyeon ()" and its three prescriptions Sinkihwan, Geumgwe-sinkihwan, and Jesaeng-sinkihwan belong to Gamibang. This study confirmed the effect of SKHGMB on renal dysfunction in an NS model induced by puromycin aminonucleoside (PAN). The experimental NS model was induced in male Sprague Dawley (SD) rats through injection of PAN (50 mg·kg-1)via the femoral vein. SKHGMB not only reduced the size of the kidneys increased due to PAN-induced NS, but also decreased proteinuria and ascites. In addition, SKHGMB significantly ameliorated creatinine clearance, creatinine, and blood urea nitrogen. SKHGMB relieved glomeruli dilation and tubules fibrosis in the glomeruli of the NS model. SKHGMB inhibited the protein and mRNA levels of the NLRP3 inflammasome including NLRP3, ASC, and pro-caspase-1 in NS rats. SKHGMB reduced the protein and mRNA levels of fibrosis regulators in NS rats. The results indicated that SKHGMB exerts protective effects against renal dysfunction by inhibiting of renal inflammation and fibrosis in NS rats.

The Modulation of Nrf-2/HO-1 Signaling Axis by Carthamus tinctorius L. Alleviates Vascular Inflammation in Human Umbilical Vein Endothelial Cells.

Carthamus tinctorius L., known as safflower, has been used in traditional treatment for cardiovascular, cerebrovascular, and diabetic vascular complications. We proposed to investigate how the ethanol extract of Carthamus tinctorius L. (ECT) can be used ethnopharmacologically and alleviate vascular inflammatory processes under cytokine stimulation in human vascular endothelial cells. Using the optimized HPLC method, six markers were simultaneously analyzed for quality control of ECT. Pretreatment with ECT (10-100 µg/mL) significantly reduced the increase of leukocyte adhesion to HUVEC by TNF-α in a dose-dependent manner. Cell adhesion molecules (CAMs) such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial cell selectin (E-selectin) are decreased by ECT. In addition, ECT significantly suppressed TNF-α-induced oxidative stress referring to reactive oxygen species (ROS) production. p65 NF-κB nuclear translocation and its activation were inhibited by ECT. Furthermore, pretreatment of ECT increased the HO-1 expression, and nuclear translocation of Nrf-2. These data suggest the potential role of ECT as a beneficial therapeutic herb in vascular inflammation via ROS/NF-kB pathway and the regulation of Nrf-2/HO-1 signaling axis is involved in its vascular protection. Thus, further study will be needed to clarify which compound is dominant for protection of vascular diseases.

Samchulkunbi-Tang Alleviates Vascular Endothelial Disorder and Renal Dysfunction in Nitric Oxide-Deficient Hypertensive Rats.

Samchulkunbi-tang (SCT, Shen Zhu Jian pi tang in Chinese) is said to have been first recorded by Zheng Zhi Zhun Sheng during the Ming Dynasty in China. Records of SCT in Korea are known to have been cited in Donguibogam (Dong Yi Bao Jian in Chinese), Uibang Hwaltu (Yi Fang Huo Tao in Chinese), and Bang Yak Hapyeon (Fang Yao He Bian in China). Although SCT is widely used in treating chronic gastritis and gastric ulcers, the beneficial effect on renal vascular function is unknown. Hypertension is a risk factor for cardiovascular disease and endothelial dysfunction in humans and experimental animal models of arterial hypertension. In addition, kidney dysfunction is characterized by hypertension diseases. This study was conducted to evaluate the effect of SCT on the vascular function in vitro (human umbilical cord endothelial cells, HUVECs) and in vivo (NG-nitro-L-arginine methyl ester, L-NAME-induced hypertensive rats). The phosphorylation of protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) is closely related to nitric oxide (NO) production in HUVECs, and SCT in this study significantly increased these. For three weeks, hypertensive rat models were induced by L-NAME administration (40 mg/kg/day) with portable water. It was followed by oral administration with 100 and 200 mg/kg/day for two weeks to confirm the effectiveness of SCT. As a result, systolic blood pressure decreased in the SCT-treated groups, compared with that in the L-NAME-induced hypertensive group. SCT treatment restored vasorelaxation by stimulating acetylcholine and cGMP production in the thoracic aorta. In addition, SCT treatment decreased intima-media thickness, attenuated the reduction of eNOS expression, and increased endothelin-1 expression. It also increased p-Akt and p-eNOS expression in hypertensive rat aorta. Furthermore, regarding renal function parameters, SCT ameliorated urine osmolality, urine albumin level, serum creatinine, and blood urea nitrogen levels. These results demonstrate that the oriental medicine SCT exerts potent vascular and renal protective effects on nitric oxide-deficient hypertensive rats and HUVECs.

Effect of Geumgwe-Sinkihwan on Renal Dysfunction in Ischemia/Reperfusion-Induced Acute Renal Failure Mice.

Renal ischemia-reperfusion (I/R) injury is an important cause of acute renal failure (ARF). Geumgwe-sinkihwan (GSH) was recorded in a traditional Chines medical book named "Bangyakhappyeon" in 1884. GSH has been used for treatment for patients with diabetes and glomerulonephritis caused by deficiency of kidney yang and insufficiency of kidney gi. Here we investigate the effects of GSH in mice model of ischemic acute kidney injury. The mice groups are as follows; sham group: C57BL6 male mice, I/R group: C57BL6 male mice with I/R surgery, GSH low group: I/R + 100 mg/kg/day GSH, and GSH high group: I/R + 300 mg/kg/day GSH. Ischemia was induced by clamping both renal arteries and reperfusion. Mice were orally given GSH (100 and 300 mg/kg/day) during 3 days after surgery. Treatment with GSH significantly ameliorated creatinine clearance, creatinine, and blood urea nitrogen levels. Treatment with GSH reduced neutrophil gelatinase associated lipocalin (NGAL) and kidney injury molecule-1 (KIM-1), specific renal injury markers. GSH also reduced the periodic acid-Schiff and picro sirius red staining intensity in kidney of I/R group. Western blot and real-time RT-qPCR analysis demonstrated that GSH decreased protein and mRNA expression levels of the inflammatory cytokines in I/R-induced ARF mice. Moreover, GSH inhibited protein and mRNA expression of inflammasome-related protein including NLRP3 (NOD-like receptor pyrin domain-containing protein 3, cryoprin), ASC (Apoptosis-associated speck-like protein containing a CARD), and caspase-1. These findings provided evidence that GSH ameliorates renal injury including metabolic dysfunction and inflammation via the inhibition of NLRP3-dependent inflammasome in I/R-induced ARF mice.

New Therapeutic Insight into the Effect of Ma Huang Tang on Blood Pressure and Renal Dysfunction in the L-NAME-Induced Hypertension.

In this study, we evaluated the effect of a traditional herbal formula, Ma Huang Tang (MHT), on blood pressure and vasodilation in a rat model of NG-nitro-L-arginine methylester- (L-NAME-) induced hypertension. We found that MHT-induced vascular relaxation in a dose-dependent manner in rat aortas pretreated with phenylephrine. However, pretreatment of endothelium-intact aortic rings with L-NAME, an inhibitor of nitric oxide synthesis (NOS), or 1H-[1, 2, 4]-oxadiazole-[4, 3-α]-quinoxalin-1-one (ODQ), an inhibitor of soluble guanylyl cyclase, significantly abolished vascular relaxation induced by MHT. MHT also increased the production of guanosine 3',5'-cyclic monophosphate (cGMP) in the aortic rings pretreated with L-NAME or ODQ. To examine the in vivo effects of MHT, Sprague Dawley rats were treated with 40 mg/kg/day L-NAME for 3 weeks, followed by administration of 50 or 100 mg/kg/day MHT for 2 weeks. MHT was found to significantly normalize systolic blood pressure and decreased intima-media thickness in aortic sections of rats treated with L-NAME compared to that of rats treated with L-NAME alone. MHT also restored the L-NAME-induced decrease in vasorelaxation response to acetylcholine and endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) expression. Furthermore, MHT promoted the recovery of renal function, as indicated by osmolality, blood urea nitrogen (BUN) levels, and creatinine clearance. These results suggest that MHT-induced relaxation in the thoracic aorta is associated with activation of the nitric oxide/cGMP pathway. Furthermore, it provides new therapeutic insights into the regulation of blood pressure and renal function in hypertensive patients.

Protective Effects of Ethanolic Extract from Rhizome of Polygoni avicularis against Renal Fibrosis and Inflammation in a Diabetic Nephropathy Model.

Progressive diabetic nephropathy (DN) in diabetes leads to major morbidity and mortality. The major pathological alterations of DN include mesangial expansion, extracellular matrix alterations, tubulointerstitial fibrosis, and glomerular sclerosis. Polygoni avicularis is widely used in traditional oriental medicine and has long been used as a diuretic, astringent, insecticide and antihypertensive. However, to the best of the authors' knowledge, the effects of the ethanolic extract from rhizome of Polygoni avicularis (ER-PA) on DN have not yet been assessed. The present study aimed to identify the effect of ER-PA on renal dysfunction, which has been implicated in DN in human renal mesangial cells and db/db mice and investigate its mechanism of action. The in vivo experiment was performed using Polygoni avicularis-ethanol soluble fraction (ER-PA) and was administrated to db/db mice at 10 and 50 mg/kg dose. For the in vitro experiments, the human renal mesangial cells were induced by high glucose (HG, 25 mM). The ER-PA group showed significant amelioration in oral glucose tolerance, and insulin resistance index. ER-PA significantly improved the albumin excretion and markedly reduced plasma creatinine, kidney injury molecule-1 and C-reactive protein. In addition, ER-PA significantly suppressed inflammatory cytokines. Histopathologically, ER-PA attenuated glomerular expansion and tubular fibrosis in db/db mice. Furthermore, ER-PA suppressed the expression of renal fibrosis biomarkers (TGF and Collagen IV). ER-PA also reduced the NLR family pyrin domain containing 3 inflammatory factor level. These results suggest that ER-PA has a protective effect against renal dysfunction through improved insulin resistance as well as the inhibition of nephritis and fibrosis in DN.

Identification of securinine as vascular protective agent targeting atherosclerosis in vascular endothelial cells, smooth muscle cells, and apolipoprotein E deficient mice.

BACKGROUND: Atherosclerosis is a chronic vascular disease and characterized by accumulation within the intima of inflammatory cells, smooth muscle cells, lipid, and connective tissue. PURPOSE: The purpose of the present study was to identify natural agents that commonly reverse advanced atherosclerotic plaque to early atherosclerotic plaque. METHODS: Differentially expressed genes (DEGs) were analyzed in silico. The differentially expressed genes from 9 intimal thickening and 8 fibrous cap atheroma tissue which were collected from GEO data were assessed by the connectivity map. Natural candidate securinine, a main compound from Securinega suffruticosa, was selected and administrated 1, 5 mg/kg/day in apolipoprotein-E-deficient (ApoE KO) mice for 18 weeks. RESULTS: Securinine significantly showed lowered blood pressure and improvement of metabolic parameters with hyperlipidemia. The impairment in vasorelaxation was remarkably decreased by treatment with securinine. H&E staining revealed that treatment with securinine reduced atherosclerotic lesions. Securinine suppressed the expression of adhesion molecules and matrix metalloproteinase-2/-9 in both ApoE KO and vascular endothelial cells (HUVEC). In HUVEC pretreatment with securinine significantly inhibited ROS generation and NF-κB activation. Growth curve assays using the real-time cell analyzer showed that securinine significantly decreased TNF-α-induced aortic smooth muscle cell proliferation and migration in a dose-dependent manner. CONCLUSION: Securinine may be a potential natural candidate for the treatment of atherosclerosis because it attenuates vascular inflammation and dysfunction as well as vascular lesion.

Betulinic Acid Protects DOX-Triggered Cardiomyocyte Hypertrophy Response through the GATA-4/Calcineurin/NFAT Pathway.

Cardiac hypertrophy is a major risk factor for heart failure and leads to cardiovascular morbidity and mortality. Doxorubicin (DOX) is regarded as one of the most potent anthracycline antibiotic agents; however, its clinical usage has some limitations because it has serious cardiotoxic side effects such as dilated cardiomyopathy and congestive heart failure. Betulinic acid (BA) is a pentacyclic-cyclic lupane-type triterpene that has been reported to have anti-bacterial, anti-inflammatory, anti-vascular neogenesis, and anti-fibrotic effects. However, there is no study about its direct effect on DOX induced cardiac hypertrophy and apoptosis. The present study aims to investigate the effect of BA on DOX-induced cardiomyocyte hypertrophy and apoptosis in vitro in H9c2 cells. The H9c2 cells were stimulated with DOX (1 µM) in the presence or absence of BA (0.1-1 µM) and incubated for 24 h. The results of the present study indicated that DOX induces the increase cell surface area and the upregulation of hypertrophy markers including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta-myosin heavy chain (ß-MHC), and Myosin Light Chain-2 (MLC2) in H9c2 cells. However, the pathological hypertrophic responses were downregulated after BA treatment. Moreover, phosphorylation of JNK, ERK, and p38 in DOX treated H9c2 cells was blocked by BA. As a result of measuring the change in ROS generation using DCF-DA, BA significantly inhibited DOX-induced the production of intracellular reactive oxygen species (ROS) when BA was treated at a concentration of over 0.1 µM. DOX-induced activation of GATA-4 and calcineurin/NFAT-3 signaling pathway were remarkably improved by pre-treating of BA to H9c2 cells. In addition, BA treatment significantly reduced DOX-induced cell apoptosis and protein expression levels of Bax and cleaved caspase-3/-9, while the expression of Bcl-2 was increased by BA. Therefore, BA can be a potential treatment for cardiomyocyte hypertrophy and apoptosis that lead to sudden heart failure.

Anti-Vascular Inflammatory Effect of Ethanol Extract from Securinega suffruticosa in Human Umbilical Vein Endothelial Cells.

Securiniga suffruticosa is known as a drug that has the effect of improving the blood circulation and relaxing muscles and tendons, thereby protects and strengthen kidney and spleen. Therefore, in this study, treatment of Securiniga suffruticosa showed protective effect of inhibiting the vascular inflammation in human umbilical vein endothelial cells (HUVECs) by inducing nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) coupling pathway. In this study, Securiniga suffruticosa suppressed TNF-α (Tumor necrosis factor-α) induced protein and mRNA levels of cell adhesion molecules such as intracellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and Interleukin-6 (IL-6). Pretreatment of HUVEC with Securiniga suffruticosa decreased the adhesion of HL-60 cells to Ox-LDL (Oxidized Low-Density-Lipoprotein)-induced HUVEC. Moreover, Securiniga suffruticosa inhibited TNF-α induced intracellular reactive oxygen species (ROS) production. Securiniga suffruticosa also inhibited phosphorylation of IκB-α in cytoplasm and translocation of NF-κB (Nuclear factor-kappa B) p65 to the nucleus. Securiniga suffruticosa increased NO production, as well increased the phosphorylation of eNOS and Akt (protein kinase B) which are related with NO production. In addition, Securiniga suffruticosa increased the protein expression of GTPCH (Guanosine triphosphate cyclohydrolase Ⅰ) and the production of BH4 in HUVEC which are related with eNOS coupling pathway. In conclusion, Securiniga suffruticosa has a protective effect against vascular inflammation and can be a potential therapeutic agent for early atherosclerosis.

Sauchinone Protects Renal Mesangial Cell Dysfunction against Angiotensin II by Improving Renal Fibrosis and Inflammation.

Abnormal and excessive growth of mesangial cells is important in the pathophysiologic processes of diabetes-associated interstitial fibrosis and glomerulosclerosis, leading to diabetic nephropathy, which eventually turns into end-stage renal disease. Sauchinone, a biologically-active lignan isolated from aerial parts of Saururus chinensis, has anti-inflammatory and anti-viral activities effects on various cell types. However, there are no studies reporting the effects of sauchinone on diabetic nephropathy. The present study aims to investigate the role of sauchinone in mesangial cell proliferation and fibrosis induced by angiotensin II, as well as the underlying mechanisms of these processes. Human renal mesangial cells were induced by angiotensin II (AngII, 10 µM) in the presence or absence of sauchinone (0.1-1 µM) and incubated for 48 h. In this study, we found that AngII induced mesangial cell proliferation, while treatment with sauchinone inhibited the cell proliferation in a dose-dependent manner. Pre-treatment with sauchinone induced down-regulation of cyclins/CDKs and up-regulation of CDK inhibitor, p21, and p27kip1 expression. In addition, AngII-enhanced expression of fibrosis biomarkers such as fibronectin, collagen IV, and connective tissue growth factor (CTGF), which was markedly attenuated by sauchinone. Sauchinone also decreased AngII-induced TGF-ß1 and Smad-2, Smad-3, and Smad-4 expression. This study further revealed that sauchinone ameliorated AngII-induced mesangial inflammation through disturbing activation of inflammatory factors, and NLRP3 inflammasome, which is composed of the NLRP3 protein, procaspase-1, and apoptosis-associated speck-like protein containing a CARD (ASC). Moreover, pretreatment of sauchinone inhibited NF-κB translocation and ROS production in AngII-exposed mesangial cells. These data suggest that sauchinone has a protective effect on renal proliferation, fibrosis and inflammation. Therefore, sauchinone might be a potential pharmacological agent in prevention of AngII-induced renal damage leading to diabetic nephropathy.

The Inhibitory Effect of Ojeoksan on Early and Advanced Atherosclerosis.

Atherosclerosis is closely related to vascular dysfunction and hypertension. Ojeoksan (OJS), originally recorded in an ancient Korean medicinal book named "Donguibogam", is a well-known, blended herbal formula. This study was carried out to investigate the beneficial effects of OJS on atherosclerosis in vitro and in vivo. Western-diet-fed apolipoprotein-E gene-deficient mice (ApoE -/-) were used for this study for 16 weeks, and their vascular dysfunction and inflammation were analyzed. OJS-treated ApoE -/- mice showed lowered blood pressure and glucose levels. The levels of metabolic parameters with hyperlipidemia attenuated following OJS administration. Hematoxylin and eosin (H&E) staining revealed that treatment with OJS reduced atherosclerotic lesions. OJS also suppressed the expression of adhesion molecules and matrix metalloproteinases (MMPs) compared to Western-diet-fed ApoE -/- mice and tumor necrosis factor-alpha (TNF-α)-stimulated human umbilical vein endothelial cells (HUVECs). Expression levels of MicroRNAs (miRNA)-10a, -126 3p were increased in OJS-fed ApoE -/- mice. OJS significantly increased the phosphorylation of endothelial nitric oxide synthase (eNOS) and protein kinase B (Akt), which are involved in nitric oxide (NO) production. OJS also regulated eNOS coupling by increasing the expression of endothelial GTP Cyclohydrolase-1 (GTPCH). Taken together, OJS has a protective effect on vascular inflammation via eNOS coupling-mediated NO production and might be a potential therapeutic agent for both early and advanced atherosclerosis.

Samul-Tang Regulates Cell Cycle and Migration of Vascular Smooth Muscle Cells against TNF-α Stimulation.

Samul-Tang (SMT), consisting of four medicinal herbs, is a well-known herbal prescription treating hematological disorders related symptoms. Our previous study demonstrated that SMT attenuated inflammation of vascular endothelial cells. In condition of retained vascular dysfunction, vascular inflammation is initiated and results in activation of smooth muscle cells (SMCs). Activated SMCs lose control of cell cycle regulation and migrate into intima, resulting in formation of atheroma. Here, we further investigated whether SMT suppresses proliferation and migration of SMCs. SMT showed antiproliferative effects on SMCs by suppressing [3H]-thymidine incorporation against TNF-α stimulation. Underlying mechanisms of antiproliferative effects were found to be resulting from cell cycle regulation. SMT downregulated expression of cyclin D1-CDK4 and cyclin E-CDK2 complexes and upregulated p21waf1/cip1 and p27kip1. SMT also suppressed migration of SMCs against TNF-α stimulation. This is thought to have resulted from suppressing MMP2 and MMP9 expressions and ROS production. In summary, SMT attenuates abnormal migration of vascular smooth muscle cells via regulating cell cycle and suppressing MMPs expression and ROS production. Our study suggests that SMT, a traditionally used herbal formula, protects vascular smooth muscle cells and might be used as an antiatherosclerotic drug.

Ligustilide attenuates vascular inflammation and activates Nrf2/HO-1 induction and, NO synthesis in HUVECs.

BACKGROUND: Ligustilide is a bioactive phthalide derivative isolated from Cnidii Rhizoma (Cnidium officinale, rhizome) and Angelicae Gigantis Radix (Angelica gigas Nakai, root) which are both medicinal herbs used to treat circulatory disorders. Vascular endothelium is a central spot in developing cardiovascular diseases and chronic vascular inflammation might result in atherosclerosis development. PURPOSE: We previously found out that a traditional herbal formula, Samul-Tang (Si-Wu-Tang, containing Cnidii Rhizoma and Angelicae Gigantis Radix), attenuated vascular inflammation in human umbilical vein endothelial cells (HUVECs). However, which compound was responsible for vascular protective action remained unclear. Here, we investigated vascular protective potential of an isolated single compound, (Z)-ligustilide. METHODS: MTT assay, western blotting, immunofluorescence, electrophoretic mobility shift assay was performed. BCECF-AM, CM-H2DCFDA, DAF-FM diacetate were used as a fluorescent indicator. RESULTS: Ligustilide suppressed HL-60 monocyte adhesion and CAMs (ICAM-1, VCAM-1, E-selectin) expression in HUVECs. Ligustilide significantly inhibited TNF-α-increased production of ROS and activated NF-κB signaling pathway. Also, ligustilide treated HUVECs exhibited significant HO-1 induction via Nrf2 nuclear translocation and endothelial NO synthesis. CONCLUSION: Present study demonstrates that ligustilde attenuates vascular inflammation and activate defense system of endothelial cell. Ligustilide is a bioactive compound which might prevent cardiovascular complications such as thrombosis or atherosclerosis.

Samchuleum attenuates diabetic renal injury through the regulation of TGF­ß/Smad signaling in human renal mesangial cells.

Renal mesangial cell proliferation is a major clinical feature of diabetic nephropathy (DN) and includes glomerulosclerosis and renal fibrosis. Samchuleum (SCE) is a traditional herbal mixture that is recorded in the ancient Korean medical book, Donguibogam. The present study attempted to determine whether SCE treatment was able to improve high glucose (HG)­induced mesangial cell fibrosis and glomerulosclerosis in primary cultured human mesangial cells. Thymidine incorporation under HG induction was increased, but was decreased by SCE in a dose dependent manner. Pretreatment with SCE led to a downregulation in the expression of cyclins and cyclin­dependent kinases (CDKs), and an upregulation of the CDK inhibitors, p21waf1/cip1 and p27kip1. In addition, SCE exposure markedly suppressed the reactive oxygen species signaling pathway under HG conditions. HG enhanced the expression levels of fibrosis­promoting mediators, including collagen IV and connective tissue growth factor, which were markedly attenuated by SCE. SCE treatment inhibited HG­induced fibronectin mRNA expression and decreased the expression of transforming growth factor (TGF)­ß1, Smad­2 and Smad­4, whereas Smad­7 expression increased under HG. SCE treatment induced the degradation of the extracellular matrix by blocking TGF­ß1/Smad signaling. Therefore, the present study suggested that the inhibitory effect of SCE on mesangial proliferation and renal fibrosis may be an effective therapy in the treatment of renal dysfunction leading to DN.

Inhibitory effect of brassinin on TNF­α­induced vascular inflammation in human umbilical vein endothelial cells.

Brassinin, a phytoalexin firstly identified as a constituent of Chinese cabbage, has been demonstrated to exhibit antiproliferative effects on various cancer cell lines, by reducing reactive oxygen species (ROS) production via regulation of the antioxidant pathway. The present study aimed to explore the protective effects of brassinin in TNF­α­induced vascular inflammation in human umbilical vein endothelial cells (HUVECs). Pretreatment with brassinin significantly inhibited adhesion of U937 cells to TNF­α­induced HUVECs in a dose­dependent manner. Brassinin treatment decreased the expression levels of cell adhesion molecules, including intracellular adhesion molecule­1 (ICAM­1), vascular cell adhesion molecule­1 (VCAM­1), and endothelial­selectin (E­selectin) following stimulation with TNF­α in HUVECs. In addition, pretreatment with brassinin decreased the protein expression levels of nuclear factor (NF)­κB p65 in the nucleus, suggesting that brassinin inhibited NF­κB p65 nuclear translocation. Brassinin treatment also markedly decreased the mRNA expression levels of interleukin­8 in a dose­dependent manner. Finally, brassinin pretreatment significantly decreased TNF­α­induced intracellular reactive oxygen species (ROS) production in HUVECs compared with control. The present results therefore suggest that brassinin may serve as a potential therapeutic agent for atherosclerosis.

Hwangryunhaedoktang exerts anti-inflammation on LPS-induced NO production by suppressing MAPK and NF- κB activation in RAW264.7 macrophages.

OBJECTIVE: This study aimed to evaluate whether Hwangryunhaedoktang (HHT), a herbal compound, has an inhibitory effect on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophages. METHODS: The effects of HHT were evaluated by confirming nitric oxide (NO) production and expression of inducible NO synthase (iNOS) and mitogen-activated protein kinases (MAPKs) in LPS-stimulated RAW264.7 macrophages via the Griess assay, Western blotting, and real-time reverse transcription quantitative polymerase chain reaction. Western blot analyses and luciferase assays were used to evaluate whether HHT has an effect on the phosphorylation and translocation of nuclear factor-κB (NF-κB). The secretion and expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined via enzyme-linked immunosorbent assay and Western blot analyses. RESULTS: HHT suppressed LPS-induced NO production and expression of iNOS in a dose-dependent manner. Additionally, MAPKs activation was also attenuated via inhibition of phosphorylation of extracellular signal-regulated kinases 1/2, c-Jun N-terminal kinase and p38 which were related to inflammatory pathway. Furthermore, HHT also effectively attenuated NF-κB activation and its translocation to the nucleus, a process that is closely linked to inflammation. LPS normally induced the expression of inflammatory cytokines such as TNF-α and IL-6, but the secretion and expression of TNF-α and IL-6 were significantly attenuated by pretreating the cells with HHT. CONCLUSION: HHT suppressed LPS-induced NO production by blocking the activation of NF-κB and MAPK signaling pathways in RAW264.7 macrophages. Furthermore, HHT may have an anti-inflammatory effect by suppressing the LPS-induced secretion of TNF-α and IL-6. Therefore, the traditional herbal formula HHT might be a useful potential therapeutic agent for inflammation.

Plantago asiatica L. Ameliorates Puromycin Aminonucleoside-Induced Nephrotic Syndrome by Suppressing Inflammation and Apoptosis.

OBJECTIVE: Nephrotic syndrome, a kidney disease with a variety of causes, is mainly characterized by heavy proteinuria, hypoproteinemia, and ascites. This study was designed to evaluate the underlying mechanism of action of Plantago asiatica L. (PAL) in treating nephrotic syndrome induced by puromycin aminonucleoside. METHODS: PAL has been used in Asia as a traditional medicine and dietary health supplement. Sprague-Dawley (SD) rats were intravenously injected with puromycin aminonucleoside (75 mg/kg/day), then treated with either Losartan (30 mg/kg/day) or PAL (200 mg/kg/day) by oral gavage for seven days. RESULTS: PAL significantly decreased ascites, proteinuria level, and plasma lipid parameters. In addition, treatment with PAL attenuated histological damage and hypoalbuminemia. Treatment with PAL also restored podocin expression and reduced inflammation markers such as intracellular adhesion molecules (ICAM-1), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor alpha (TNF-α) and high-mobility group box-1 (HMGB1). Lower expression levels of the apoptosis markers Bax, caspase-3 and capase-9 were documented in SD rats receiving PAL. PAL also significantly decreased the phosphorylation levels of MAPKs such as ERK, JNK and p38. CONCLUSION: As a multifunctional agent, PAL has a renoprotective effect in nephrotic syndrome rat models. The anti-inflammatory and anti-apoptotic properties, along with reductions in hyperlipidemia and ascites, represent important therapeutic effects. These results indicate that Plantago asiatica is likely to be a promising agent in the treatment of nephrotic syndrome.

Wiryeongtang regulates hypertonicity-induced expression of aquaporin-2 water channels in mIMCD-3 cells.

The kidneys have a key role in the homeostasis of water excretion and reabsorption. Water channels, particularly aquaporin-2 (AQP2), are important proteins in water homeostasis in the body through the short­term and long-term regulation of water permeability. Wiryeongtang (WRT) is a well-known traditional oriental medicine, which is used for the treatment of chronic edema and dysuresia. The aim of the present study was to evaluate the inhibitory effect of WRT on the hypertonicity-induced expression of AQP2 in the inner medullary collecting duct cell line (IMCD­3). Western blotting, reverse transcription­polymerase chain reaction and immunofluorescence analysis were performed to determine the effect of WRT under hypertonic stress. WRT attenuated the 175 mM NaCl hypertonic stress­induced increases in protein and mRNA levels of AQP2 and apical membrane insertion in a concentration­dependent manner. However, no differences were observed in the levels of AQP1, AQP3 or AQP4 between the hypertonic stress and WRT groups. WRT attenuated the hypertonicity-induced phosphorylation of glucocorticoid-inducible protein kinase 1. In addition, the mRNA expression of tonicity­responsive enhancer binding protein was attenuated by WRT under hypertonic stress. Pretreatment with WRT also decreased the hypertonic stress­induced expression of AQP2, as with KT5720, a protein kinase A inhibitor. These results provided evidence of the beneficial effect of the traditional formula WRT in regulating water balance in hypertonic stress of the renal collecting ducts.